Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mtor-dependent translation initiation

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1 Am J Physiol Endocrinol Metab 293: E1416 E1425, First published September 11, 2007; doi: /ajpendo Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mtor-dependent translation initiation Renán A. Orellana, 1,2 Asumthia Jeyapalan, 2 Jeffery Escobar, 1 Jason W. Frank, 1 Hanh V. Nguyen, 1 Agus Suryawan, 1 and Teresa A. Davis 1 1 United States Department of Agriculture/Agricultural Research Service Children s Nutrition Research Center, and 2 Critical Care Section, Department of Pediatrics, Baylor College of Medicine, Houston, Texas Submitted 5 March 2007; accepted in final form 30 August 2007 Orellana RA, Jeyapalan A, Escobar J, Frank JW, Nguyen HV, Suryawan A, Davis TA. Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mtor-dependent translation initiation. Am J Physiol Endocrinol Metab 293: E1416 E1425, First published September 11, 2007; doi: /ajpendo In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 g kg 1 h 1 ), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mtor, 4E-BP1, and S6K1 phosphorylation and eif4g-eif4e association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mtor-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis. growth; sepsis; mammalian target of rapamycin; eukaryotic initiation factor 4G; ribosomal protein S6 kinase THE SYSTEMIC INFLAMMATORY RESPONSE and sepsis syndromes elicited in the host in response to a bacterial pathogen or its products (35) are associated with hypermetabolism, increased oxygen consumption and energy expenditure, activation of peripheral protein catabolism, and alteration in the flux of metabolic substrates between skeletal muscle and liver (4, 39). Derangements in skeletal muscle glucose and amino acid metabolism evoked by sepsis result from the body s attempt to maintain the balance between peripheral substrate stores and the increased metabolic demands required to activate the immune system and the stress response (4, 37). These adaptative responses are different from those in the normal fasting person Address for reprint requests and other correspondence: R. A. Orellana, USDA/ARS Children s Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Rm. 9057, Houston, TX ( orellana@bcm. edu). in that the immune response is the drive for the metabolic enhancement (11, 59). In an attempt to establish homeostasis, amino acids are released from peripheral tissues and shifted to the liver for promotion of hepatic protein synthesis, gluconeogenesis, and urea synthesis (24, 25). Plasma amino acid concentrations in patients with sepsis or disease are lower than in persons without sepsis (17, 30, 49), and attempts have been made to use the extent of this difference as a marker of the severity and prognosis of the disease process (16, 58). The uptake of amino acids by the liver is elevated, despite the reduced plasma amino acid concentration; thus, hepatic extraction of serum amino acids is increased (25). As a consequence, the metabolic clearance and turnover of endogenously released amino acids is enhanced (16). The healthy neonatal pig maintains elevated rates of protein synthesis in skeletal muscle as a consequence of a high sensitivity and responsiveness to stimulation of protein synthesis by both insulin and amino acids (12, 13), which accelerates the rate of translation of mrna through activation of eukaryotic translation initiation factors (14, 50). Conversely, in the liver of normal neonatal pigs, protein synthesis is stimulated by amino acids, but not by insulin (43). The translation of mrna into protein occurs in three distinct phases: initiation, elongation, and termination (41), and the regulation of the translation process occurs during initiation and elongation (53). An early event in translation initiation is the formation the 43S preinitiation complex, by the association of the eukaryotic initiation factor (eif)2, the initiator methionyl-trna (met-trna i ), and a molecule of GTP to the 40S ribosomal subunit. A second event occurs through activation of a signaling pathway involving the mammalian target of rapamycin (mtor), which allows the 43S preinitiation complex to bind to capped mrna. This process is mediated by the phosphorylation of eif4e-binding protein-1 (4E-BP1) and 70-kDa ribosomal protein S6 kinase (S6K1). 4E-BP1 is a repressor protein that competes with eif4g for binding to eif4e. The availability of eif4e is dependent on decreased affinity of eif4e for 4E-BP1 when 4E-BP1 is phosphorylated. Phosphorylation and availability of eif4e increase the association of eif4e with eif4g and the binding of mrna to the 43S ribosomal complex (26). The phosphorylation of S6K1 has been proposed to enhance translation of mrnas that have tracts of oligopyrimidines in their 5 end (TOP mrnas) and to enhance global rates of protein synthesis (52). The elongation process occurs after translation initiation and requires that eukaryotic elongation factor-2 The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. E /07 $8.00 Copyright 2007 the American Physiological Society

2 (eef2) couples to GTP hydrolysis (41), and thus the phosphorylation of eef2 is associated with decreased elongation (50). In contrast to septic mature rats, which exhibit a profound reduction in muscle protein synthesis (56), endotoxin [lipopolysaccharide (LPS)]-infused neonatal pigs present only a moderate decrease in skeletal muscle protein synthesis when insulin and amino acids are maintained in the fed range (48), and the reduction in translation initiation appears more profound than the reduction in protein synthesis, suggesting that different anabolic mechanisms regulate protein synthesis in skeletal muscle at the translational level (29, 45). In LPSinfused neonatal pigs, the response of muscle protein synthesis to insulin stimulation is maintained largely independently of amino acid stimulation and despite persistent suppression of translation initiation (45 47), suggesting that insulin accelerates protein synthesis in muscle through an mtor-independent process (46, 47). Our previous studies in LPS-infused neonatal pigs suggest that the response of muscle protein synthesis to insulin may be mediated by the elongation step in peptide formation (46). Conversely, LPS stimulates protein synthesis by enhancing translation initiation in the liver, and insulin does not affect this response (46, 47). Therefore, we hypothesized that amino acids stimulate protein synthesis in skeletal muscle and liver of neonatal pigs during acute endotoxemia independently of insulin stimulation. To address this hypothesis, we infused neonatal pigs with the Escherichia coli (E. coli) endotoxin lipopolysaccharide (LPS), while blood glucose and plasma insulin concentrations were maintained at fasting levels and plasma amino acid concentrations were clamped at either fasting or fed levels. Fractional protein synthesis rates were then determined in muscles of different fiber types, and they were compared with those in the liver and other visceral organs. Translation initiation-signaling proteins were examined in longissimus dorsi (LD) and liver. The results show that, during acute endotoxemia, muscle protein synthesis in neonates retains the ability to respond to amino acid stimulation by enhancing translation initiation but liver protein synthesis is not further enhanced by provision of amino acids. METHODS E1417 Animals. Three cross-bred (Landrace Yorkshire Hampshire Duroc) pregnant sows (Agriculture HQ, Texas Dept. of Criminal Justice, Huntsville, TX) were housed in lactation crates, provided with water ad libitum, and received a commercial diet (no. 5084; PMI Feeds, Richmond, IN) for 1 2 wk before farrowing. After farrowing, the piglets resided with the sow, and 3 days prior to the experiment they were anesthetized for sterile catheter insertion into a jugular vein and a carotid artery. Piglets were then returned to the sow and allowed to suckle freely until studied. The protocol was approved by the Animal Care and Use Committee of Baylor College of Medicine and was conducted in accordance with the National Research Council s Guide for the Care and Use of Laboratory Animals. Experimental design. Thirty-four piglets (5 6 days of age; kg body wt) were assigned randomly to control (n 20) or LPS (n 14) treatment groups. After h of fasting, each animal was placed in a sling restraint system to perform pancreatic glucose-amino acid clamps (Fig. 1). Following previously described techniques, the average basal concentrations of whole blood glucose and plasma branched-chain amino acids (BCAA) were obtained (47). Real-time plasma BCAA concentrations were determined by rapid enzymatic kinetic assay (2) to establish the average basal concentration of BCAA to be used in the subsequent clamp procedure (Fig. 1). The clamp was initiated with a primed (20 g/kg), continuous somatostatin (Bachem, Torrance, CA) infusion at 100 g kg 1 h 1 to block endogenous insulin secretion ( 1 h). A physiological replacement glucagon infusion (150 ng kg 1 h 1 ; Eli Lilly, Indianapolis, IN) was provided 10 min after the initiation of somatostatin and was continued to the end of the clamp period. Simultaneously with the glucagon infusion, insulin was infused at 7 ng kg 0.66 min 1 to maintain plasma insulin concentrations of 3 5 U/ml to simulate a fasting insulinemic state. Glucose and amino acids were clamped to the individual basal fasting levels during the infusion by monitoring the blood glucose and plasma BCAA at 5- to 10-min intervals and adjusting the infusion rates of dextrose and a balanced amino acid mixture to maintain plasma BCAA and blood glucose within 10% of the baseline fasting level (Fig. 1). The amino acid mixture is based on the amino acid composition of body protein (13) and contained (in mmol/l) arginine (20.1), histidine (12.9), isoleucine (28.6), leucine (34.3), lysine (27.4), methionine (10.1), phenylalanine (12.1), threonine (21.0), tryptophan (4.4), valine (34.1), alanine (27.3, 38% provided as alanyl-glutamine), aspartate (12.0), cysteine (6.2), glutamate (23.8), glutamine (17.1, Fig. 1. Schematic representation of endotoxemic pancreatic substrate clamps and tracer infusion indicating times of hormonal infusion, tracer administration, and substrate clamp.

3 E % provided as alanyl-glutamine), glycine (54.3, 4% provided as glycyltyrosine), proline (34.8), serine (23.8), taurine (2.0), and tyrosine (7.2, 83% provided as glycyl-tyrosine). One hour after the initiation of the somatostatin infusion, the LPS group received a continuous infusion (10 g kg 1 h 1 )ofe. coli endotoxin (lyophilized E. coli Serotype 0111-B4, Sigma Chemical, St. Louis, MO) that was continued for 8 h while the control group received an equal volume of sterile normal saline solution (Fig. 1). Blood samples were obtained at 1-h intervals for later determination of circulating insulin and individual essential and nonessential amino acid concentrations. After 6 h of LPS/saline infusion (6 t), the infusion rate of the balanced amino acid mixture was adjusted to clamp the plasma BCAA either to replicate fasting ( 500 nmol BCAA/ml; Control, n 9; LPS, n 6) or fed ( 1,000 nmol BCAA/ml; Control AA, n 11; LPS AA, n 8) levels and to maintain the plasma BCAA concentration within 10% of the desired level (13). Blood glucose continued in the targeted fasting range in both groups. All animals were euthanized 8 h after the LPS infusion began (8t), i.e., 2 h after increasing the amino acid infusion rate to the fed level (6t), thereby providing sufficient time to achieve a steady state for the targeted circulating glucose, amino acids, and insulin levels before the animals were subjected to a flooding dose of the tracer (Fig. 1). Measuring protein synthesis in vivo. Seven hours and 30 min after the LPS infusion was initiated (Fig. 1), tissue fractional rates of protein synthesis were measured in vivo using the flooding dose technique (1.5 mmol/kg body wt, equal to 1 mci/kg body wt) of L-[4-3 H]phenylalanine (Amersham Biosciences, Piscataway, NJ) as previously described (13). Eight hours after the LPS infusion began (Fig. 1), the animals were euthanized with an intravenous dose of pentobarbital sodium (50 mg/kg body wt). LD, gastrocnemius, masseter, diaphragm, and cardiac muscles and liver, stomach, jejunum, lung, pancreas, and kidney were rapidly removed, frozen in liquid nitrogen, and stored at 70 C until analysis. Frozen tissues were then processed as previously described (13, 21). The fractional rate of protein synthesis (K s), the percent of protein mass synthesized in a day, was calculated as K s (%/day) [(S B/S A) (1,440/t)] 100, where S B is the specific radioactivity of the protein-bound phenylalanine, S A is the mean specific radioactivity of the tissue free phenylalanine during the labeling period determined from the amount at the time of tissue collection, corrected by linear regression of the change in blood specific radioactivity against time, and t is the time of labeling in minutes. Plasma hormones and substrate assays. Whole blood glucose, plasma insulin, and total BCAA concentrations were determined as previously described (2, 14, 47). Whole body net glucose disposal rates (mg glucose kg 1 min 1 ) and whole body net amino acid disposal rates (mmol kg 1 h 1 ) were determined from the infusion rates of dextrose and amino acid solution needed to maintain baseline fasting levels of blood glucose and plasma amino acids, respectively. Individual plasma amino acid concentrations were measured with an HPLC method (PICO-TAG reverse-phase column; Waters, Milford, MA) as previously described (13). A commercially available kit was used to measure plasma urea nitrogen (PUN; Biotron Diagnostics, Hemet, CA). Protein immunoblot analysis. LD and liver tissue homogenates were separated on polyacrylamide gel electrophoresis. Samples were run at the same time in triple-wide gels (C.B.S. Scientific C, Del Mar, CA) for each assay to eliminate interassay variation (14, 18). Proteins were electrophoretically transferred to polyvinylidene difluoride transfer membranes (Bio-Rad, Hercules, CA), which were incubated with the correct primary antibodies, washed, and exposed to a proper secondary antibody as previously described (18). Immunoblots with antiphosphospecific antibodies were stripped in stripping buffer (Pierce Biotechnology, Rockford, IL) and reprobed with the corresponding non-phosphospecific antibodies for normalization. Blots were developed using an enhanced chemiluminescence kit (Amersham Life Sciences, Arlington Heights, IL), visualized, and analyzed using a ChemiDoc-It Imaging System (UVP, Upland, CA). The antibodies used in the immunoblotting process were mtor (total and Ser 2448 ; Cell Signaling), S6K1 (total and Thr 389 ; Cell Signaling), 4E-BP1 (total; Bethyl Laboratories, Montgomery, TX; Thr 70, Cell Signaling), and eef2 phosphorylation (total and Thr 56 ; Cell Signaling) (18). Quantification of eif4e eif4g complexes. These complexes were immunoprecipitated using an anti-eif4e monoclonal antibody (gift of Dr. Leonard Jefferson, Penn State University College of Medicine, Hershey, PA) from aliquots of fresh tissue homogenates. Briefly, samples were homogenized in 7 volumes of buffer (in mm: 20 HEPES, 2 EGTA, 50 NaF, 100 KCl, and 0.2 EDTA, ph 7.4) containing Sigma P3840 Protease Inhibitor Cocktail (Sigma Chemical) and centrifuged at 10,000 g for 10 min at 4 C. Supernatants were incubated overnight at 4 C with constant rocking with anti-eif4e antibody (18). Immunoprecipitates were recovered with goat antimouse IgG magnetic beads (Polysciences, Warrington, PA), washed, and resuspended in sample buffer as previously described (18) and immediately subjected to protein immunoblot analysis using rabbit anti-eif4g (Bethyl Laboratories) antibody. Amounts of eif4g were corrected by the eif4e recovered from the immunoprecipitate (18). Statistical analyses. By use of statistical software (Minitab for Windows), analysis of variance (general linear modeling) was used to assess the effect of LPS, amino acids, and their interaction on protein synthesis and translation initiation factor activation. Student s t-test was used to test for differences between groups. To determine the effectiveness of the clamp procedure, individual amino acid, glucose, and insulin concentrations and translation initiation factor activation in each treatment group were compared with their basal concentrations by use of t-tests. Probability values of 0.05 were considered statistically significant for all comparisons. Data are presented as means SE. RESULTS Metabolic response to LPS during pancreatic substrate clamps. LPS-infused animals presented higher temperature values in response to endotoxin infusion (P 0.05; Table 1). Baseline fasting plasma insulin levels were obtained for each individual animal, and those fasting levels were maintained during the experimental period in control animals (P 0.05; Table 1). LPS induced a modest but statistically significant elevation of insulin levels compared with controls in both the fasting and fed amino acids groups (P 0.05; Table 1). In control and LPS-infused animals, whole blood glucose was maintained at fasting levels during the entire experimentation (P 0.05; Table 1). Baseline plasma BCAA levels were obtained for each individual animal at the beginning of the experiment, and those plasma levels were maintained during the entire experimental period in both fasting control and LPS-infused animals (P 0.05; Table 1). In the amino acid-stimulated group, BCAA levels similar to those observed in the fed state ( 1,000 nmol/ml) were obtained by infusing the balanced amino acid mixture to both control and LPSinfused animals during the last 2 h of LPS infusion. The fed BCAA levels obtained were different from those in the baseline and in the fasting condition in both control and LPSinfused animals (P 0.05; Table 1). Fed BCAA levels were not different between groups (P 0.05; Table 1). Baseline PUN was similar in all animals during fasting. In the presence of fed amino acid levels, PUN levels were lower in LPSinfused animals compared with controls (P 0.05; Table 1). Net whole body glucose disposal rates increased in response to LPS, but not in response to the amino acid infusion (P

4 Table 1. Plasma insulin, glucose, BCAA, and PUN concentration in LPS and control pigs during endotoxemic pancreatic substrate clamp Markers Control LPS Temperature, o F * Insulin, U/ml Baseline Fasting AA levels * Fed AA levels * Glucose, mg/dl Baseline Fasting AA levels Fed AA levels BCAA, nmol/ml Baseline Fasting AA levels Fed AA levels 1, , PUN, mg/dl Baseline Fasting AA levels Fed AA levels * Values are means SE; Control, n 9; LPS, n 6; Control AA, n 11; LPS AA, n 8/group. AA, amino acids; BCAA, branched-chain AA; PUN, plasma urea nitrogen. Insulin was targeted to fasting levels (2-5 U/ml); fed AA groups were provided a balanced AA mixture to achieve fed AA levels ( 1,000 nmol BCAA/ml). Significantly different from baseline; *significantly different between control and LPS; significantly different between fast vs. fed (P 0.05). E1419 Although the total plasma concentrations of BCAA were similar in control and LPS-infused animals, LPS decreased leucine concentrations during fasting (P 0.05; Table 2). Leucine, isoleucine, and valine in the LPS-infused animals were raised in plasma to levels similar to those in controls when a balanced amino acid solution was infused to achieve fed amino acid concentrations in both groups (P 0.05). The concentrations of aromatic amino acids (phenylalanine, tyrosine, and tryptophan) were not affected by LPS and were raised to fed levels in response to the amino acid infusion (P 0.05). LPS raised the plasma concentration of histidine during fasting and fed states in LPS-infused animals compared with controls (P 0.05). Plasma concentrations of arginine were reduced by LPS in the fasting state and remained at lower concentrations when amino acids were raised to fed levels (P 0.05). Threonine concentrations were reduced by LPS (P 0.05) and were not raised significantly in response to amino acids (P 0.05). The concentrations of the nonessential amino acids glutamine, glycine, proline, and serine were higher in LPS-infused animals only after amino acids were infused (P 0.05). The circulating concentrations of asparagine, citrulline, glycine, methionine, ornithine, lysine, and taurine were raised in response to amino acids and were not affected by LPS (P 0.05). 0.05; Fig. 2A). Both amino acid and LPS infusions increased the whole body amino acid disposal rates (P 0.05; Fig. 2B). We did not find an interaction between the effects of the LPS and amino acid infusions on whole body amino acid disposal rates, which suggests that those effects were additive (P 0.05; Fig. 2B). Fig. 2. Net whole body glucose (A) and amino acid (AA) disposal (B) rates in LPS-infused animals compared with controls during endotoxemic pancreatic substrate clamps. Fed AA groups were provided a balanced AA mixture to achieve fed AA levels ( 1,000 nmol BCAA/ml), whereas glucose and insulin were maintained at fasting levels. LPS, but not AA, increased glucose disposal rates (P 0.05). AA disposal rates increased in response to LPS and AA infusion (P 0.05). a,b,c,d Values with different letters differ (P 0.05). Table 2. AA concentrations in LPS and control pigs during endotoxemic pancreatic substrate clamps AA Control Control AA LPS LPS AA EAA Arginine * * Histidine * * Isoleucine * * Leucine * * Lysine * * Methionine * * Phenylalanine * Threonine Tryptophan * * Valine * NEAA Alanine * * Asparagine * * Citrulline * * Glutamine * * Glycine ,034 66* ,487 57* Ornithine * * Proline * * Serine * * Taurine * * Tyrosine * * EAA 1, , * , * NEAA 1, , * 1, , * BCAA ,311 76* ,160 33* Total AA 2, , * 2, , * Values are means SE in nmol/ml; Control, n 9; LPS, n 6; Control AA, n 11; LPS AA, n 8/group. EAA and NEAA, essential and nonessential AA. Individual plasma AA concentrations were measured with an HPLC method. Fed AA groups were provided a balanced AA mixture to achieve fed AA levels ( 1,000 nmol BCAA/ml) while glucose and insulin were maintained at fasting levels. *Different between fast and fed AA; different between control and LPS (P 0.05).

5 E1420 Protein synthesis in muscles during endotoxemic pancreatic substrate clamps. In the presence of amino acid levels similar to those seen in fasting, LPS reduced protein synthesis in the LD ( 22%, P 0.05; Fig. 3) and gastrocnemius ( 31%, P 0.05), increased protein synthesis in the diaphragm ( 35%, P 0.05), and had no significant effect on masseter and heart muscles (P 0.05). Increasing amino acids to fed levels accelerated muscle protein synthesis rates in the LD (controls by 75%; LPS by 65%, P 0.05), gastrocnemius (controls by 40%, LPS by 68%, P 0.05), masseter (controls by 61%, LPS by 47%, P 0.05), and diaphragm (controls by 41%, P 0.05; LPS by 26%, P 0.06) muscles. Neither LPS nor amino acids stimulated protein synthesis in heart muscle of control and LPS-infused animals (P 0.05). There was no interaction between LPS and amino acids with regard to the effect on muscle protein synthesis, indicating that the effects of LPS on muscle protein synthesis were independent of those of the amino acid infusion (P 0.05). Protein synthesis in viscera during endotoxemic pancreatic substrate clamps. In the presence of fasting conditions, LPS stimulated protein synthesis in the liver ( 31%), lung ( 34%), spleen ( 39%), pancreas ( 44%), and kidney ( 30%) compared with controls (P 0.05; Fig. 4, A and B). Raising amino acids to fed levels in control pigs increased protein synthesis in the liver ( 35%) and pancreas ( 52%, P 0.05). Amino acids further enhanced the effect of LPS in the pancreas ( 25%) but not in other tissues of LPS-treated animals (P 0.05). The effects of LPS on protein synthesis in the lung, spleen, pancreas, and kidney were independent of those of the amino acid infusion (P 0.05). We found an interaction between LPS and amino acids on protein synthesis in liver, indicating that the effects of LPS on liver protein synthesis can be influenced by amino acid infusion (P 0.05). Neither LPS nor amino acids influenced protein synthesis in the jejunum or stomach (P 0.05). Effects of amino acids on translation initiation in muscle and liver in response to LPS during pancreatic substrate clamps. In muscle, in the presence of amino acid concentrations similar to those seen in fasting, LPS reduced the protein synthetic efficiency, i.e., the total protein synthesized per total RNA (P 0.05; Fig. 5A). In both LPS-infused and control animals, Fig. 4. Fractional protein synthesis rates in liver, lung, spleen, pancreas (A) and jejunum, stomach, and kidney (B) in LPS and control pigs during endotoxemic pancreatic substrate clamps. Values are means SE; Control, n 9; LPS, n 6, Control AA, n 11; LPS AA, n 8/group. Insulin was targeted to fasting levels (2 4 U/ml); fed AA groups were provided a balance amino acid mixture to achieve fed AA levels ( 1,000 nmol BCAA/ ml). a,b,c Values with different letters differ (P 0.05). raising amino acids to fed levels enhanced the protein synthetic efficiency in muscle (P 0.05; Fig. 5A). The protein synthetic capacity of the tissue, measured by the RNA-to-protein ratio, was not affected by LPS or amino acids in muscle (P 0.05; data not shown). In liver, both LPS and amino acids enhanced the protein synthetic efficiency (P 0.05; Fig. 5B), but the protein synthetic capacity of the tissue was not affected by LPS or amino acids in liver (P 0.05; data not shown). Protein synthetic efficiency did not increase further in the presence of amino acids in LPS-infused animals (P 0.05; Fig. 5B). In the presence of fasting conditions, LPS decreased eif4geif4e association in muscle (Fig. 5D). When amino acids were raised to levels seen during the postprandial state, mtor, 4E-BP1, and S6K1 phosphorylation and eif4g-eif4e association increased in muscle of both control and LPS-treated animals (P 0.05; Figs. 5 and 6). Amino acids did not alter eef2 phosphorylation in muscle of control and LPS-infused animals (P 0.05; Fig. 6E). In liver, both amino acids and LPS increased 4E-BP1 and S6K1 phosphorylation, but only amino acids reduced eef2 phosphorylation (P 0.05; Fig. 6). DISCUSSION Fig. 3. Fractional protein synthesis rates in longissimus dorsi (LD), gastrocnemius, masseter, heart, and diaphragm muscles in LPS and control pigs during endotoxemic pancreatic substrate clamps. Values are means SE; Control, n 9; LPS, n 6, Control AA, n 11; LPS AA, n 8/group. Insulin was targeted to fasting levels (2 4 U/ml); fed AA groups were provided a balanced AA mixture to achieve fed AA levels ( 1,000 nmol BCAA/ml). a,b,c,d Values with different letters differ (P 0.05). Muscle catabolism has been recognized as a critical feature in the metabolic dysregulation that accompanies systemic inflammation and sepsis (10, 39). The effects of critical illness on protein metabolism have been compared with autocannibalism, in which the organism utilizes its own nitrogen stores to supply the amino acids required to maintain the acute-phase response, gluconeogenesis, healing, and repair (8, 24, 38). Controversy exists as to whether whole body nitrogen balance can be maintained by supplying exogenous amino acids in animals

6 E1421 Fig. 5. Effects of AA on protein synthetic efficiencies in LD (A) and liver (B), mtor phosphorylation in LD (C), and eukaryotic initiation factor (eif)4g-eif4e association in LD (D) of LPS and control pigs during endotoxemic pancreatic substrate clamps. Phosphorylated forms were normalized to total content. Values are means SE; Control, n 9; LPS, n 6, Control AA, n 11; LPS AA, n 8/group. Insulin was targeted to fasting levels (2 4 U/ml); fed AA groups were provided a balanced AA mixture to achieve fed AA levels ( 1,000 nmol BCAA/ml). a,b,c,d Values with different letters differ (P 0.05). and humans with sepsis (15, 44). Most of the studies have determined the nitrogen-sparing effect of amino acids at the whole body level, and it has been suggested that leucine, more than other amino acids, exerts a critical role in maintaining protein balance (15, 61). However, some essential and nonessential amino acids may become limiting during anabolic conditions (18) or sepsis (36), and resistance to stimulation by leucine has been found in vitro and in vivo during sepsis (34, 61). In adult rats subjected to experimental sepsis, supplementing a balanced amino acid solution has been shown to enhance protein synthesis rates in skeletal muscle (27). In healthy neonatal animals, muscle protein synthesis exhibits a robust response to amino acid stimulation, and this response decreases with development (14). In the present report, we have demonstrated that the muscle of neonatal pigs retains the ability to respond to the stimulatory action of a balanced amino acid solution by increasing protein synthesis after LPS administration and that this response is modulated by the activation of factors that regulate mrna binding to the ribosomal complex. However, provision of amino acids does not further enhance the increase in liver protein synthesis induced by endotoxemia. Effects of endotoxemia in neonatal animals. The acute inflammatory response and the metabolic consequences elicited by endotoxin infusion in both adult and neonatal swine have been validated in prior publications and include a septic-like response, an increase in insulin levels, increased amino acid disposal, and a decrease in muscle protein synthesis rates (20, 47, 48). In the current study, insulin levels were slightly but significantly higher in the LPS group compared with controls despite provision of a somatostatin infusion that we had previously shown to block endogenous insulin secretion (43, 47). This suggests that either the somatostatin dose was less effective in the presence of LPS or that insulin clearance also was impaired in response to a LPS challenge in this study (31). In developing rats, a diminished response to somatostatin has been reported (63). It is possible that the difference in insulin concentrations could affect the stimulatory effect of amino acids on muscle protein synthesis. However, even though insulin levels were higher in LPS-infused animals compared with controls at their parallel amino acid level, e.g., fasting and fed states, the LPS-associated reduction in baseline muscle protein synthesis rates was still demonstrated at both the fasting and fed amino acid levels. Although neonatal animals appear to have similar cytokine and hormonal reactions in response to endotoxin stimulation compared with mature swine (20, 48), lack of maturation of the metabolic response may affect gluconeogenesis, protein metabolism, and substrate utilization (22, 47, 63). Therefore, when facing an acute catabolic insult such as endotoxin infusion, neonatal animals may present with hypoglycemia (63) as a consequence of the metabolic alterations required to sustain the metabolic needs of immune activation during acute stress (44), such as impaired hormonal interactions, higher insulin levels, preservation of insulin sensitivity, and diminished glucose availability. However, the neonatal animal appears capable of preserving the anabolic response of muscle protein synthesis to insulin and amino acids when challenged by the sepsis-induced alterations in metabolism (48). Effects of LPS on whole body glucose disposal. During the acute inflammatory response, whole body glucose uptake is increased to sustain metabolism in inflammatory cells, to support the higher metabolic rate induced by thermogenesis, and to provide enough glucose to different organs (44). Some stress hormones (such as cortisol, glucagon, and epinephrine) increase the glucose supply by enhancing glycogenolysis and gluconeogenesis, promoting insulin resistance, and altering insulin homeostasis (59, 60). Previously, we (47) demonstrated that LPS has an additive effect to the insulin-related increase in net whole body glucose disposal in neonatal pigs. In the present study, net whole body glucose uptake increased in

7 E1422 Fig. 6. Effects of AA on eif4e-binding protein-1 (4E-BP1) phosphorylation at Thr 70 in LD (A) and liver (B), 70-kDa ribosomal protein S6 kinase (S6K1) phosphorylation at Thr 389 in LD (C) and liver (D), and eukaryotic elongation factor (eef)2 phosphorylation at Thr 56 in LD (E) and liver (F) of LPS and control pigs during endotoxemic pancreatic substrate clamps. Phosphorylated forms were normalized to total content. Values are means SE; Control, n 9; LPS, n 6, Control AA, n 11; LPS AA, n 8/group. Insulin was targeted to fasting levels (2 4 U/ml); fed AA groups were provided a balanced amino acid mixture to achieve fed AA levels ( 1,000 nmol BCAA/ ml). a,b,d Values with different letters differ (P 0.05). response to LPS infusion, and higher infusion rates of dextrose were required to maintain baseline fasting blood glucose levels compared with fasting controls. Since net whole body glucose disposal rates were not affected by amino acid infusion, we speculate that, when glucose is provided to maintain fasting blood glucose levels, the amino acids provided in our pancreatic substrate clamp are not utilized for gluconeogenesis during acute LPS infusion in neonatal pigs (40). Whole body amino acid disposal and plasma amino acid profile in response to insulin during LPS infusion. During acute inflammation and sepsis, the balance between whole body amino acid uptake and plasma amino acid availability depends on a catabolic response that mobilizes amino acids from muscle for the synthesis of hepatic acute-phase reactants and gluconeogenesis (4, 16, 25). Some amino acids may retain a specific role during sepsis, such as arginine as a substrate for the synthesis of nitric oxide (44, 61), alanine as a gluconeogenic substrate (62), and leucine as a regulator of protein anabolism in muscle (15, 44, 61). In the current study, net whole body amino acid disposal was augmented in neonatal pigs in response to LPS and amino acid infusion, and this response was additive. Since liver protein synthesis increased in response to LPS infusion in our model, we hypothesize that the increase of whole body amino acid disposal rates in response to LPS infusion occurs, at least in part, as a consequence of increased amino acid uptake in the liver for the synthesis of acute-phase reactants (4). In neonatal pigs, this LPS-induced increase in whole body amino acid disposal rates is independent of and additive to the balanced amino acid supplementation necessary to achieve fed amino acid levels that allow an increase in muscle protein synthesis during acute endotoxemia. Besides their role composing tissue structure, amino acids have been shown to play an important role in regulating human metabolism in normal conditions (42) and during sepsis (44). Amino acid profiles change during illness (30, 58, 61), but their relation to disease severity is not clear (57, 58). BCAA have been shown to spare protein synthesis during sepsis (27), but their role in counteracting catabolism during sepsis remains controversial (15, 57). Plasma BCAA levels decrease in response to endotoxin in swine (4), and in our study the LPSinfused neonatal pigs required higher amino acid infusion rates to maintain baseline fasting plasma BCAA. More specifically, leucine has been shown to play a regulatory role in anabolism in normal conditions (54) and during sepsis (15, 32, 61). In our study, leucine levels remained lower in LPS-infused animals during fasting despite an increase in amino acid disposal rates and near-maintenance of baseline fasting BCAA concentrations, similar to reports in adult swine (4). All plasma BCAA (i.e., leucine, isoleucine, and valine) in the LPS-infused animals were raised to fed control amino acid levels when a balanced amino acid solution was provided to raise amino acid levels to that of the fed state. This event suggests that during acute inflammation the utilization of leucine is driven by the acute inflammatory response, and compensation for the deficit in leucine can be nearly attained by providing a balanced amino acid mixture. The increase in plasma leucine is accompanied by an increase in muscle protein synthesis in neonatal pigs. Contrary to reports in children and adults with sepsis and infection (7, 30, 49), we did not find alterations in aromatic amino acids (i.e., phenylalanine tyrosine, and tryptophan). In the present study in neonatal pigs, alanine, an amino acid that is important as a gluconeogenic substrate and important nitrogen carrier during catabolic states (62), was increased during LPS infusion, similar to previous reports in adult swine challenged acutely with endotoxin (4). In our study, plasma alanine levels in LPS-infused pigs remained higher when amino acids were provided to achieve fed amino acid levels and dextrose was provided to maintain fasting glucose levels, suggesting that the alanine release from muscle was not affected and therefore proteolysis continued despite adequate amino acid supply. This also suggests that alanine released from muscle may exceed alanine whole body utilization during acute endotoxemia in neonatal pigs (4). Arginine plasma concentrations were reduced by LPS in the fasting and fed states despite amino acid supplementation. Arginine utilization has been reported to be altered in septic children (1), and it is an important precursor of nitric oxide during acute inflammation (44). Furthermore, the increased concentrations of proline, glycine, and histidine when amino acids were provided to

8 achieve a fed state likely reveal an imbalance in amino acid metabolic pathways during sepsis or immaturity of such pathways in the neonatal animal (6). Effects of amino acids on protein synthesis in muscle during LPS infusion. During sepsis, whole body amino acid uptake may not reflect the metabolic needs of each individual organ, because protein metabolism differs depending on the role of that organ during the systemic inflammatory response (5, 48). Systemic inflammation targets skeletal muscle and induces muscle catabolism (11) and a reduction in muscle protein synthesis rates (33, 48). This effect occurs in muscles that contain primarily glycolytic muscle fibers, such as the gastrocnemius and LD muscles (56). In the present study, LPS reduced skeletal muscle protein synthesis in the LD and gastrocnemius muscles of neonatal pigs in the presence of fasting amino acids but did not affect the masseter and heart muscles, which are predominantly composed of fibers of oxidative metabolism. LPS administration also increased protein synthesis in the diaphragm, a unique muscle of mixed fiber composition, probably secondary to the associated tachypnea induced by LPS infusion (48). Raising plasma amino acids to levels seen in the fed state augmented protein synthesis rates in the LD, gastrocnemius, masseter, and diaphragm, but not heart, muscles in both control and LPS-infused animals. It is possible that the effect of LPS on protein synthesis in the diaphragm and the lack of effect of LPS on protein synthesis in the heart muscle could be attributable to either the effect of continuous autonomic contraction or that their fibers share similar metabolic properties with the masseter muscle. Although muscle protein synthesis rates increased in response to amino acids in both groups, the LPS-infused animals presented a lower baseline during fasting amino acid levels than controls. This suggests that, even though the ability of muscle protein synthesis to respond to amino acids is maintained during an acute LPS infusion in neonatal pigs, acute endotoxemia reduces the rate of muscle protein synthesis achieved during the provision of amino acids. Since the decrease in muscle protein synthesis occurs simultaneously with a decrease in plasma leucine levels and increases in response to achievement of plasma amino acid levels similar to those seen during the fed state, it appears that protein synthesis in skeletal muscle responds to proper maintenance of plasma amino acid levels, including leucine, in septic neonates. Effects of amino acids on protein synthesis in viscera during LPS infusion. Despite the reduction in skeletal muscle protein synthesis that occurs during sepsis, it has been demonstrated that whole body protein synthesis is elevated as a result of increased visceral tissue protein synthesis (3, 55). In the current study, as in our previous studies (13, 43), amino acids increased protein synthesis in the liver and pancreas of healthy neonatal pigs (43). LPS increased protein synthesis in the liver, lung, spleen, pancreas, and kidney during fasting compared with controls, similar to previous reports (4, 47, 48). The stimulation of liver protein synthesis during sepsis reflects the synthesis of acute-phase reactants (4, 16). This is further supported by a decreased concentration in threonine in the LPS-infused animals despite amino acid supplementation, as threonine has been associated with increased utilization for acute-phase reactant synthesis and maintenance of gut integrity during sepsis and inflammation (3, 19, 51). Although we did not find an increase in protein synthesis in the jejunum in the E1423 present study, there have been reports of increased intestinal protein synthesis during acute inflammation (3, 48), possibly due to the immune activation of the intestinal mucosa and mucin production (19). In addition, the increases in protein synthesis in other visceral tissue may be related to activation of immunogenic tissues, i.e., lung and spleen (3), and organspecific synthetic processes, i.e., LPS-induced synthesis of renal arginine (23). Largely, the results suggest that the increase in protein synthesis in most visceral tissues during endotoxemia cannot be further enhanced by amino acid administration. Since the PUN remained lower in amino acid-infused LPS animals compared with amino acid-infused controls, we hypothesize that the amino acids infused in endotoxemic animals were utilized for anabolic process in skeletal muscle. In our study, both amino acids and LPS increased protein synthesis in the liver, but provision of amino acids did not further enhance the LPS-induced increase in liver protein synthesis. It is possible that providing a different balance of amino acids may prevent futile recycling of amino acids and improve the efficiency of nitrogen utilization during neonatal sepsis. Effects of amino acids and LPS on translation initiation. Previously, we (46) demonstrated that insulin stimulates muscle protein synthesis in skeletal muscle of LPS-infused neonatal pigs despite suppression of insulin-stimulated mtordependent translation initiation signaling and that insulin appears to modulate protein synthesis in muscle of neonates during endotoxemia by modulating the elongation process. In the current study, LPS reduced the protein synthetic efficiency in muscle, but raising amino acids to fed levels enhanced the protein synthetic efficiency. These amino acid-induced changes were associated with activation of mtor-dependent translation initiation signaling, as evidenced by the increased mtor, 4E-BP1, and S6K1 phosphorylation, and increased formation of the active eif4g eif4e complex in both control and LPS-infused animals. However, amino acids did not alter the elongation process. This finding suggests that insulin and amino acids increase muscle protein synthesis in neonatal endotoxemia by stimulating different translational mechanisms. In our study, we found that eif4g eif4e complex formation was decreased in response to LPS in animals in the presence of fasting amino acid concentrations, in parallel with the decrease in protein synthesis in skeletal muscle. This suggests that the LPS-induced decrease in protein synthesis rates in skeletal muscle in the absence of insulin and amino acid stimulation may occur via inhibition of the eif4 group of translation initiation factors, and this event might limit the restoration of fractional protein synthesis rates by insulin and/or amino acid stimulation in the presence of LPS. Further studies are required to determine the mechanisms that regulate the LPS-induced decrease in baseline protein synthesis rates, which may include a redistribution of ribosomal mrna into non-polysome fractions (28) or cleavage of initiation factors (9). We previously showed that LPS enhances translation initiation in liver (46). In the present study, both LPS and amino acids enhanced protein synthetic efficiency in the liver, but the effects of amino acids and LPS were not additive. Those changes paralleled an increase in 4E-BP1 and S6K1 phosphorylation in both control and LPS-infused animals and reduced eef2 phosphorylation in controls only, in response to amino acids. These results, together with the failure of amino acids to

9 E1424 further enhance liver protein synthesis in LPS-infused animals, suggest that both translation initiation and fractional synthesis rates in the liver have been maximally stimulated by LPS and no that further additive effect may be achieved by amino acid supplementation. Perspectives. We have demonstrated that protein synthesis in skeletal muscle responds to amino acid stimulation during short-term endotoxemia. Furthermore, this response in skeletal muscle contrasts with the failure of visceral tissue in young septic animals to further enhance protein synthesis in response to amino acid stimulation. The increase in muscle protein synthesis in LPS-infused pigs in response to fed amino acid concentrations is opposed by a decrease in baseline muscle protein synthesis rates. The results of the present study support the hypothesis that muscle protein synthesis in neonates retains a robust response to amino acid stimulation during acute endotoxemia despite a decrease in the baseline rates of protein synthesis that occurs in skeletal muscle during fasting. In contrast to the mechanisms that regulate insulin stimulation during neonatal endotoxemia, amino acids stimulate muscle protein synthesis by enhancing mtor-dependent translation initiation signaling. These findings suggest that amino acid supplementation may help alleviate the catabolic response triggered by sepsis in neonatal muscle. ACKNOWLEDGMENTS We thank Marta Fiorotto for helpful comments, Jerome Stubblefield for assistance with care of animals, William Liu, Jillian Fleming, and Marie Ng for laboratory assistance, and Linda Weiser for secretarial assistance. GRANTS This work is a publication of the US Department of Agriculture, Agricultural Research Service (USDA/ARS) Children s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children s Hospital, Houston, Texas. This project has been funded, in part, by National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Mentored Award K08 AR This research was also supported in part by National Institutes of Health Grants K12 HD (R. A. Orellana) and R01 AR (T. A. Davis) and the USDA/ARS under Cooperative Agreement no.r (T. A. Davis). The contents of this publication do not necessarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. REFERENCES 1. Argaman Z, Young VR, Noviski N, Castillo-Rosas L, Lu XM, Zurakowski D, Cooper M, Davison C, Tharakan JF, Ajami A, Castillo L. 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